This document provides instructions for routine alignment and operation of a JEOL 1200-EX II TEM for an independent user. It describes starting conditions, high voltage generation and conditioning, electron beam generation, illumination system alignment including gun alignment, condenser lens alignment, condenser stigmation, condenser aperture centering, and sample insertion. Basic usage concepts like focusing and beam centering are also covered, along with the digital imaging system and diffraction mode.
This document provides instructions for routine alignment and operation of a JEOL 1200-EX II TEM for an independent user. It describes starting conditions, high voltage generation and conditioning, electron beam generation, illumination system alignment including gun alignment, condenser lens alignment, condenser stigmation, condenser aperture centering, and sample insertion. Basic usage concepts like focusing and beam centering are also covered, along with the digital imaging system and diffraction mode.
This document provides instructions for routine alignment and operation of a JEOL 1200-EX II TEM for an independent user. It describes starting conditions, high voltage generation and conditioning, electron beam generation, illumination system alignment including gun alignment, condenser lens alignment, condenser stigmation, condenser aperture centering, and sample insertion. Basic usage concepts like focusing and beam centering are also covered, along with the digital imaging system and diffraction mode.
TEM Components/Controls/Functions Table 2 Routine Alignment and Operation for the Independent User Starting Conditions 4 High Voltage Generation and Conditioning 4 Electron Beam Generation Preliminary 4 Gun Alignment 5 Condenser Lens Alignment 5 Condenser 2 Stigmation 5 Condenser Aperture Centering 6 Sample Insertion 6 Electron Beam Generation for Sample Observation 7 Stage Centration 7 Tilt Axis (Z Height) Alignment 7 Image Wobbler Balance 8 Voltage Center Alignment 8 Inserting and Centering the Objective Aperture 8 Objective Lens Astigmatism Compensation 9 Basic Usage Basic Concepts 10 Focusing 10 Beam and Histogram Centering 11 Shutdown 11 Digital Imaging System Basic Operation 12 Shading Correction 13 Taking Your Work With You 14 Multiple Image Alignment 15 Diffraction 16
Nils Hasselmo Hall EM Lab 6/29/2007 2
Components of the TEM Column Component Console Control* Function Illumination System Electron Gun (Filament, Wehnelt, Anode) FILAMENT knob (LL); BIAS toggles (LL) Electron source Gun Alignment Coils (gun shift and gun tilt) GUN SHIFT button and DEF X and Y knobs (LR). GUN TILT button and DEF X and Y knobs (LR). Translates and tilts the beam beneath the gun to maximize illumination Condenser Lens 1 SPOT SIZE toggle (UL) Determines smallest illumination spot size on specimen Condenser Lens 2 BRIGHTNESS knob (UL) Varies amount of illumination on specimen Condenser Stigmator COND STIG button (UL) and DEF X and Y knobs (UL and UR). Corrects for astigmatism in the condenser lens Condenser 2 Aperture Controls amount/coherence of illumination striking the specimen; reduces spherical aberration in convergent beam mode Deflector Coils SHIFT X and Y knobs (UL and UR). Translates the entire illumination system onto screen center Focus Wobbler IMAGE WOBBLER button (UR) Aids with low magnification focusing by alternating current to the beam deflector coils Specimen Manipulation System Specimen Holder Goniometer Tilts the specimen around the X-axis; Shifts the specimen vertically Specimen Selector Selects specimen grid 1 or 2 Imaging System Objective Lens FOCUS knobs (UR) Forms, magnifies, and focuses first image Objective Aperture Signal selection; Controls contrast and spherical/chromatic aberration Objective Stigmator OBJ STIG button (UL) and DEF X and Y knobs (UL and UR). Corrects for astigmatism in the objective lens Intermediate Lens Magnification SELECTOR toggle (UR) Magnifies image form objective lens and focuses diffraction pattern Intermediate (Diffraction, Field Limiting) Aperture Selects area to be diffracted Projector Lenses Magnification SELECTOR toggle (UR) Magnifies image * LL=lower left panel; UL=upper left panel; UR=upper right panel; LR=lower right drawer
Nils Hasselmo Hall EM Lab 6/29/2007 3 Routine Alignment and Operation for Independent User
Symbolism
Blue: Console Objects: For ex, FILAMENT knob Orange: Column Objects: For ex, Field Limiting Aperture Black: Imaging Program Objects: For ex, Viewport Manager window Bold Black: States objects can exist in. For ex, IN Processes used to change the states of objects. For ex, Center the beam
Starting Conditions:
The N 2 Cylinder pressure should be 500 psi or higher; the regulated N 2 gauge should read between 5 and 8 psi. Contact the staff if readings deviate from these values.
Liquid Nitrogen Anticontaminator. Add LN 2 carefully to anticontaminator dewar. Use funnel to avoid splashing. Top off after about 10 minutes.
FILAMENT knob OFF. PENNING GAUGE reading at the bottom end of the Green Zone HT button ON. The green READY lamp LIT Sample Holder IN the column. Condenser, Objective, and Field Limiting Apertures OUT (aperture assembly levers turned to the right).
If you will be using 60, 80, or 100 kV and the microscope is currently at a different kV: Slowly toggle the ACCEL VOLTAGE switch to the desired kV. Proceed to Electron Beam Generation.
If you will be using 120 kV and the microscope is at a lower kV: Proceed to High Voltage Generation and Conditioning.
Nils Hasselmo Hall EM Lab 6/29/2007 4 Alignment:
High Voltage Generation and Conditioning.
1. Slowly toggle the ACCEL VOLTAGE switch to 100 kV.
2. HT WOBBLER ON. 3. SLOWLY toggle ACCEL VOLTAGE up, stepwise, to 120 kV. Keep an eye on the PENNING GAUGE watch for wobbling and allow the needle to bottom out before your next stepwise increase. At the same time watch the BEAM CURRENT meter. Dont let the Dark Current exceed 90 uA. If it jumps to 90 or more, slow down your stepwise increase.
4. Reduce ACCEL VOLTAGE from 120 to 100 kV. TheHT WOBBLER should be left ON.
5. Wedge the H.T Conditioning Stick to push in and hold the HT COND button. This will raise the dark current about 10% and allow High Tension to go 10% above the value you are setting with the ACCEL VOLTAGE selector.
6. VERY SLOWLY repeat step 4, above. Allow the PENNING GAUGE to reach its lowest value with each kV increase Your goal now is to temporarily overvoltage the system, up to about 130-132 kV.
7. When you reach 120 kV, leave it there 5 minutes, and then reduce back down to 100 kV. Decreasing kV can be conducted at a more rapid rate; however, be conservative.
8. Release the HT COND button by removing the stick and turn HT WOBBLER OFF.
9. SLOWLY toggle the ACCEL VOLTAGE to the value you will be using.
Electron Beam Generation
1. Toggle the Magnification SELECTOR to 2500X. Set SPOT SIZE at 1. 2. SLOWLY saturate the FILAMENT knob. When saturated, the beam at crossover will show no filament structure. Use the FINE BIAS MODE toggle to adjust the BEAM CURRENT reading so that it is 10 uA more than the Dark Current reading for the accelerating voltage you are using.
3. Center and spread the beam: Focus the beam to crossover (smallest spot) with the BRIGHTNESS knob. The magnitude of knob increments can be increased by activating the STEP button. Center the beam using the SHIFT X and Y knobs. Knob increments can again be controlled with the STEP button (UL). Spread the beam with the BRIGHTNESS knob (spreading the beam is accomplished by turning the BRIGHTNESS knob COUNTERCLOCKWISE. In the underfocus direction).
Nils Hasselmo Hall EM Lab 6/29/2007 5 ILLUMINATION SYSTEM ALIGNMENT (at the beginning of a session)
Gun Alignment
1. Toggle the Magnification SELECTOR to 5000X. Toggle SPOT SIZE to 1. 2. Bring beam to crossover and center it.
3. Turn FILAMENT knob counterclockwise to create cats eye image. 4. DEFLECTOR-GUN TILT button ON -- LIT. Use DEF X and Y knobs to attain symmetry in the filament image.
5. DEFLECTOR-GUN SHIFT button ON -- LIT. Use DEF X and Y knobs to center the filament image on viewing screen. DEFLECTOR-GUN SHIFT button OFF
6. Turn the FILAMENT knob to saturation.
Condenser Lens (Spot Size) Alignment
1. Magnification SELECTOR at 5000X 2. Toggle SPOT SIZE to 5. Bring beam to crossover and center with SHIFT X and Y knobs.
3. Toggle SPOT SIZE to 1. Bring beam to crossover and center by turning the DEFLECTOR- GUN SHIFT button ON LIT and using the DEF X and Y knobs.
4. Repeat steps 2-3 until the amount of beam shift when varying SPOT SIZE is minimized.
5. DEFLECTOR-GUN SHIFT button OFF. 6. From this point on, center the beam spot with the SHIFT X and Y knobs.
Condenser 2 Stigmation
1. Toggle the Magnification SELECTOR to 10,000X Toggle SPOT SIZE to 5. 2. Bring beam to crossover and center.
3. Turn FILAMENT knob counterclockwise to produce cats eye image of filament tip. Adjust BRIGHTNESS to sharpen filament image.
4. COND STIG button ON LIT. Use DEF X and Y knobs controls to focus filament image as sharply as possible. Use the small viewing screen and the binoculars to aid with focus.
5. Turn the FILAMENT knob to saturation. 6. COND STIG button OFF.
Nils Hasselmo Hall EM Lab 6/29/2007 6 7. Check condenser astigmatism compensation by turning BRIGHTNESS knob from crossover to fully counterclockwise. Beam should be round or nearly so.
8. Toggle to the SPOT SIZE you wish to use (usually 2)
Condenser Aperture Centering
1. Insert the Condenser Aperture (lever all the way to the left). Rotate dial to select the desired aperture size (Usually #2 [200 uM]). Line up the white and black dots on the aperture assembly.
2. Center and spread the beam. If it spreads asymmetrically, it indicates that the Condenser Aperture is not well centered. Center the illumination, and thus the aperture, with the two black adjusting knobs on the Condenser Aperture assembly end and side.
3. Check that the beam is centered and ensure that the Condenser Aperture is centered once again.
Sample Insertion.
1. Slowly turn FILAMENT knob OFF.
2. Loading the Sample Holder: DO NOT touch the Sample Holder anywhere between the O-ring and the tip; the oils on your skin will contaminate the column vacuum. Use a pair of forceps to open and gently close the grid holders. The sample position nearest the tip of the dual sample holder is designated #1. Check the Sample Holder O-ring. It must be free of lint fibers or other contamination.
3. Insert the Sample Holder into the sample port with the alignment screw at 9:00 oclock position. Push and wait for airlock pump to start. After the Connector Box Lamp goes ON (red) and OFF twice, turn the sample holder fully clockwise and gently let the vacuum pull it all the way in. Do this carefully; otherwise you could damage the sample holders tip bearing if you insert the holder too fast. Use your thumb as a brake block against the sample port.
4. If you have a vacuum leak upon sample insertion and the High Tension turns off (the BEAM CURRENT meter will register no current): keep the Sample Holder IN; ensure that FILAMENT knob is OFF; allow the PENNING GAUGE to reach its lowest level; and reengage the HT (High Tension) button.
5. Adjust the sample position with the Sample Select wheel (left side of the microscope column) to select sample 1 or 2. Use the upper set of marks as the true positions.
Nils Hasselmo Hall EM Lab 6/29/2007 7 Electron Beam Generation for Sample Observation
1. SLOWLY saturate the FILAMENT.
2. Center and spread the beam. If beam isnt seen, a grid bar may be blocking it. Adjust Specimen Shifting Wheels to find it.
3. Toggle the Magnification SELECTOR to a magnification that allows you to locate an area of interest in your sampleusually 4000 5000X.
4. Use OBJ FOCUS to focus your sample. Note that at this point your image will lack contrast because the objective aperture is not inserted.
IMAGE FORMATION SYSTEM ALIGNMENT (ideally for each sample)
Stage Centration
1. Press PAGE key to display PAGE 2. 2. Adjust Specimen Shifting Wheels to X = 0, Y = 0. 3. LOW MAG button ON -- LIT. Objective Aperture OUT. 4. Toggle the Magnification SELECTOR to 150X and adjust BRIGHTNESS knob until grid is visible.
5. Adjust Sample Select Wheel until you approximately center the grid image.
Tilt Axis (Z Height) Alignment
1. Stage Tilt at ZERO degrees. 2. LOW MAG button ON LIT. Objective Aperture OUT. 3. Toggle the Magnification SELECTOR to 500X and focus your sample.
4. Disengage Stage Motor drive unit (push away).
5. Rotate the Stage Tilt (+) 20 degrees. (CCW) 6. Use the Specimen Shifting Y Wheel to locate and center on screen a conspicuous image (grid corner, piece of dirt, etc).
7. Rotate the Stage Tilt (-) 20 degrees. (CW) 8. Operate the Z-Shift knob to move the image to a point about way between where it ended up in step 6 and the screen center.
9. Repeat steps 5-8 until the image position no longer changes when the Stage Tilt is rotated +/- 20 degrees.
Nils Hasselmo Hall EM Lab 6/29/2007 8 10. Repeat steps 5-8 at 10,000 X and 30,000. At these higher magnifications, again only adjust the object position by 1/4 to 1/3 when returning toward the center spot between tilts.
11. Return Stage Tilt to ZERO degrees. Re-engage Stage Motor drive unit (pull toward you).
Image Wobbler Balance
1. Toggle the Magnification SELECTOR to 10,000X and obtain TRUE FOCUS (minimum contrast). Use the small viewing screen and the binoculars to aid with focus.
2. Bring beam to crossover and center it.
3. IMAGE WOBBLER button ON. Adjust the IMAGE WOBBLER X and Y knobs to merge separate spots into one and to minimize movement. IMAGE WOBBLER button (UR) OFF.
4. Re-Center and spread the beam.
Voltage Center Alignment
1. Toggle the Magnification SELECTOR to 60,000X. Keep beam centered as you increase your magnification.
2. Locate a distinctive feature (hole) in sample and move it to screen center. Obtain TRUE FOCUS (minimum contrast). Use the small viewing screen and the binoculars to aid with focus.
3. HT WOBBLER button ON LIT. 4. BRIGHT TILT button ON LIT. Use DEF X and Y knobs controls to minimize movement in the X and Y plane. .
5. HT WOBBLER button OFF. BRIGHT TILT button OFF.
Inserting and Centering the Objective Aperture
1. Ensure that your sample is in the beam path. 2. Press the DIFF button.
3. Use the IOS/DIFF knob to obtain the sharpest/smallest central diffraction spot. Activate the DEFLECTOR-PROJ button and use the DEF X and Y knobs to center the central diffraction spot
4. Insert the Objective Aperture (lever all the way to the left). Select the desired aperture size by lining up the white and black dots on the aperture assembly.
5. Center the Objective Aperture around the central diffraction spot with the two black adjusting knobs on the assembly end and side.
6. Press MAG 1 to get out of diffraction mode
Nils Hasselmo Hall EM Lab 6/29/2007 9 Objective Lens Astigmatism Compensation
1. Toggle the Magnification SELECTOR to 75,000 to 125,000X. Keep the beam centered as you increase your magnification. Locate a hole.
2. Use OBJ FOCUS to achieve slight overfocus (easily visible dark Fresnel fringe around the hole). At this point you may want to image using the flat panel monitors and AnalySIS software. See Digital Imaging System section
3. Adjust OBJ FOCUS in the underfocus (CCW) direction until the overfocus fringe just disappears at two opposing sides of the hole.
4. OBJ. STIG button ON -- LIT. Use DEF X and Y knobs controls to make the overfocus fringe uniform around the hole. It will become thinner as you do this.
5. Repeat steps 3-4, as many times as necessary. The amount you will turn toward underfocus will become less and less, until finally it will become very difficult to see any overfocus fringe at all.
6. When you have eliminated asymmetry in the overfocus Fresnel fringe, and can make it disappear entirely as you adjust toward underfocus, the edge of the hole will appear soft and lacking contrast -- you are now seeing True Focus. Adjustment to slight underfocus will produce a thin, uniform white Fresnel fringe inside the hole and the background film granularity will be enhanced -- this is the focus setting at which you should acquire your images
7. Alternatively, you can compensate for astigmatism by selecting an area of the holey carbon film that shows uniform density (no holes) and ensuring that the FFT is spherical at a high magnification (150,000 to 200,000X).
8. OBJ. STIG button OFF.
Nils Hasselmo Hall EM Lab 6/29/2007 10
General Usage:
Basic Concepts The goal of biological TEM is to obtain well focused images that: show high resolution; display a wide range of grey-scale values; and have adequate/uniform illumination/brightness. The Table below summarizes the main parameters a user can manipulate and their effects on these image properties.
Accelerating Voltages of 60 kV and 80 kV are appropriate for biological TEM
Sample Mass (Z) is dependent on the nature of the specimen and its staining; Sample Thickness derives from the ultramicrotome cutting thickness.
The Objective Aperture on the J EOL 1200 has settings of 50, 100, and 150 microns. The 50 micron setting is best able to produce the contrast desired for biological samples.
A Spot Size of 2 is commonly used on this TEM for biological imaging. Generally speaking, use the smallest spot size possible consistent with adequate illumination, in order to increase beam coherence and thus improve resolution. However, if you find you have to focus BRIGHTNESS nearly to crossover for adequate illumination to record an image, you should use a larger spot size.
Focusing
Unlike an SEM which is parfocal, the focus for a TEM must be adjusted every time there is a change in magnification. Use the small pop-up screen and binoculars to aid with focusing. Ensure the binoculars are in sharp focus for your eyes (when the black dot in the viewing field is in sharp focus).
Low Magnification Focusing (up to 20 kX): IMAGE WOBBLER button ON -- LIT. Use the OBJ FOCUS knob to minimize object movement in the image. The microscope is set to automatically apply a small amount of underfocus to your image once you subsequently disengage this feature.
High Magnification Focusing: There are two approaches you can use to focus at high magnification. One is to observe the behavior of fine particle detail. When this background granularity is just underfocused, it becomes sharp to the eye. The other approach is to use Fresnel fringes associated with holes/objects in the specimen which show discrete edges/boundaries. Overfocus will be seen as dark fringes; underfocus appears as white fringes; true focus will reveal no interference lines. Remember that you want a small amount of underfocus.
Nils Hasselmo Hall EM Lab 6/29/2007 11 Beam and Histogram Centering
Even with the microscope well aligned, you may need to ensure even illumination by centering the beam when you change magnification. Get in the habit of centering and spreading the beam as you make changes in magnification (especially large changes). Care must be taken in beam centering, as delicate sections are likely to be destroyed at cross-over. Likewise, beam brightness will change with changes in magnification. Activating the ZOOM button will help to keep the histogram distribution centered. Additional histogram centering is accomplished with the BRIGHTNESS knob.
Shut Down
1) MAG 1 button ON LIT and Magnification SELECTOR to 7500X 2) Toggle SPOT SIZE to 2. 3) Beam centered and spread. 4) FILAMENT knob OFF. 5) Condenser, Objective, and Field Limiting Apertures OUT (levers all the way to the left).
6) Camera OUT. 7) Remove the Sample Holder, remove your sample from it, and return the holder to the microscope.
8) HT button ON. 9) MegaView Camera Control Unit OFF; exit iTEM computer program; Computer and Flat panel displays OFF (if you are the last user of the day).
10) Turn down the microscope CRT display down. 11) Fill in all requested information in the logbook and log out of your session on the computer
Nils Hasselmo Hall EM Lab 6/29/2007 12 Digital Imaging System
Basic Operation:
1. MegaView Camera Control Unit ON. (It is found on the box on the top of left console of the TEM.) Camera out: the green lamp will light on the In/Out Hand Switch unit.
2. Select User to log into the computer.
3. Select iTEM icon on desktop to load anaySIS program. 4. Select DUAL button under Viewport Manager window and highlight the first open Image Buffer.
5. Insert the camera by pressing the green lamp on the In/Out Hand Switch. 6. Left click on camera of choice.
Camera 1 Camera 2 Camera 3 Name Snapshot Search (1) Diffraction Resolution 1376 x 1032 * 688 x 516 688 x 516 Exposure Time 2001000 ms ** 50 ms 50 ms Image Acquisition Yes No Yes Live Histogram; Live Overlay Yes Yes Yes Offset & Gain Shading Correction Yes No Yes Function Final focus; High resolution image capture *** Identifying areas of interest; Initial focus Diffraction: image acquisition***
* 1376 x 1032 is the largest resolution available for our camera setup. ** To change the Exposure Time for Camera 1: Choose Set Input under the Image pull down menu. Highlight the Camera labeled Snapshot and left click the Configure Input button on the top of the dialogue box. Select the Input Tab and adjust the Exposure Time. *** Shading Corrections MUST be made for the Cameras used for image acquisition. This will require the acquisition of an Offset and Gain Image
Nils Hasselmo Hall EM Lab 6/29/2007 13 ixed and es 7. Live image comes up on left screen. Right screen will show previous image (may be black if first capture coming up).
8. Adjust histogram to 50% (Range=45-55%, with wide spread of distribution) with BRIGHTNESS knob.
There is a Fixed Scaling and Auto Scaling button at the bottom of the Histogram window. Auto Scaling is fine for most applications. Selecting F Scaling with the blue/red sliders all the way left/right will ensure maximum information capture. Histogram stretching gamma adjustment of the captured imag should be done subsequently with programs such as Photoshop and ImageJ .
9. Left click the Acquisition Button (Button 1, 2, or 3) corresponding to the camera chosen. If you take a snapshot of Camera 1 (high resolution), you will be prompted to enter the magnification. Doing so will ensure a micron marker in your image. The size and format of the micron marker can be adjusted in the Properties dialogue box within Scalebar under the Image pull down menu.
10. If you wish to save the snapshot, highlight the corresponding buffer and do: File>Save As. Save your images in your own folder in the My Documents folder on the desktop (This folder is a shortcut to: C Document and Settings Characterization Fac My Documents. You will need to know this location for FTPing your documentssee below). If you wish to save your file as an 8 bit rather than 12 bit: When you select Save as from the File pull down menu there is an Options button. Select that button and check Convert 12 bit images to 8 bit
11. Click and highlight the next open Image Buffer will turn blue. Then click on one of the camera icons to return to live imaging.
Shading Correction
Acquiring the Offset image: FILAMENT current OFF; Sample Holder OUT of the column. Camera IN the column. Cover the viewing window of the microscope. Activate the Camera of choice by selecting the Camera icon followed by its Acquisition button. Select Acquire Reference Images under the Image pull down menu. Click the Acquire button adjacent to Offset Image.
Acquiring the Gain image: FILAMENT saturated; Sample Holder OUT of the column. Ensure the beam is centered and spread. Cover the viewing window of the microscope. Camera IN the column. Activate the Camera of choice by selecting the Camera icon followed by its Acquisition button. Select Camera Control under the Image pull down menu and ensure that the Exposure Time value there has the same value as it does within Configure Input for
Nils Hasselmo Hall EM Lab 6/29/2007 14 the camera. Select Acquire Reference Images under the Image pull down menu. Adjust BRIGHTNESS so that the histogram median is 50%. Click the Acquire button adjacent to Gain Image (Do not select Automatic intensity adjustment).
Checking the reference images: Sample Holder must still be out of the column. Ensure the beam is centered and spread. Select the Camera for which you acquired the Gain and Offset image followed by its Acquisition button. The only thing you should be able to see is homogeneous noise.
Taking Your Work With You
Burn a CD: 1. Insert CD 2. Select the file/folder of interest 3. Select Copy this File/Folder under File and Folder Tasks. 4. Select the DVD/CD Drive and click Copy. 5. You will be prompted with a You have files waiting to be copied balloon. Click on the balloon. 6. Select the File/Folder of interest in the dialogue box which appears and click Copy to CD
FTP to Server: 1. Open FileZilla on the desktop 2. Select Site Manager under the File menu. Select CFServer and enter your username and password. 3. Select and drag the file/folder of interest from the Local Site to the Remote Site.
Nils Hasselmo Hall EM Lab 6/29/2007 15 Multiple Image Alignment
1. Select the sample area and magnification of interest. In order to create a composite image you will be increasing your magnification and dividing the area of interest into subimages that will later be aligned. The subimages will be displayed in a matrix of rows and columns. You can have up to six rows and six columns. Obviously, the larger the number of subimages, the higher your magnification increase will need to be. The following example shows the numbering system for a 2 x 3 matrix. 1 2 4 3 5 6
2. Select/highlight the first open buffer. Choose Camera 2 and position the sample so that you are viewing subimage 1. Choose Camera 1 to acquire a high resolution image in the buffer (histogram median should be at 50%).
3. Select/highlight the next open buffer. Choose Camera 2 and position the sample so that you are viewing subimage 2. Ensure an overlap of about 20% with subimage 1 so that the computer program can pattern match and subsequently align the two subimages. Continue this process through the final subimage.
4. Select/highlight the first buffer subimage captured 5. In the Image pull down menu select Acquire Multiple Image and then Arrange.
6. Set up the horizontal and vertical parameters to match the matrix of subimages you just captured.
7. Make sure that Meander and Horizontal are selected. The highest Correlation quality is a value of 1
8. Click OK. An Arrange Multiple Images dialogue box with the composite image will appear
9. Select side by side as the Play with Overlap Area Options. To reposition a subimage left click, hold and move with the mouse. Checking Original Size will increase the screen magnification and allow for fine tuned alignment.
10. Checking Equalize will align the grayscale values of the composite subimages 11. Selecting Cut Margins will eliminate image borders that jut out as well as black areas. 12. Click OK and the composite image should be saved to the next open buffer where it can be saved in the usual way
Nils Hasselmo Hall EM Lab 6/29/2007 16 Diffraction
Selected Area Electron Diffraction 1. Locate sample area of interest. 2. Turn DIFF on and set camera length to 250 cm. 3. Use the IOS/DIFF knob to obtain the sharpest spot 4. Activate the DEFLECTOR-PROJ button and use the DEF X and Y knobs to center the spot. 5. Turn SAM/ROCK on. 6. Select desired magnification and field of view 7. Insert the Field Limiting Aperture and center (if image disappears, decrease magnification) 8. Use the IOS/DIFF knob to focus the Field Limiting Aperture. 9. Center the Field Limiting Aperture. 10. Remove the Objective Aperture. 11. Turn DIFF on and set camera length to 10-250 cm. 12. Use the IOS/DIFF knob to focus the diffraction pattern. 13. Insert the beam stopper. 14. Capture the image (need sections on digital and film)
Microbeam Electron Diffraction 1. Locate sample area of interest. 2. Turn DIFF on and set camera length to 250 cm. 3. Turn MAG 1 on. Set desired Magnification. 4. Center precisely the feature of interest and focus. 5. Remove Objective Aperture. 6. Select smallest Condenser Aperture. 7. Minimize Spot Size. 8. Bring beam to crossover and center. 9. Turn DIFF on and set camera length to 10 cm. 10. Turn the IOS/DIFF knob to focus diffraction pattern. 11. Insert the beam stopper 12. Capture the image (need sections on digital and film).